ABSTRACT: Biofilm formation is a key aspect of Streptococcus pneumoniae (Spn) pathogenesis. Spn in biofilms have been detected in diverse anatomical sites and shown to contribute to Spn transmission and virulence. Despite this, the environmental cues and regulatory pathways responsible for Spn biofilm formation remain unknown. Our recent studies have shown that streptococcal pyruvate oxidase (SpxB), the enzyme that converts pyruvate to acetyl phosphate and in doing so produces hydrogen peroxide (H2O2), is required for Spn biofilm formation. The goal of this project is to determine if Spn biofilm formation is the downstream result of a metabolic profile associated with excess acetyl phosphate, the result of a transcriptional response to H2O2, or a combination of both. We will: AIM 1. Determine if acetyl phosphate is required for Spn biofilm formation. Acetyl phosphate is converted to acetyl-CoA which then serves as substrate for acetylated capsular polysaccharide types. In similar fashion, we hypothesize that the production of key biofilm ECM components depends on the availability of acetyl phosphate/acetyl-CoA. We will test this by examining the biofilm-forming ability of mutant bacteria unable to produce acetyl-CoA, determining the impact of exogenous acetate and acetyl-CoA on wildtype and mutant bacteria, and identifying the metabolic changes that occur during in vitro biofilm formation under distinct acetyl- phosphate level growth conditions. AIM 2. Determine if H2O2 is a quorum signal for Spn biofilm formation. Spn has a two-component signal transduction system, CiaRH, which is responsive to oxygen and is required for biofilm formation. Based upon findings that show the oxidative state of CiaRH impacts its activity within oral streptococci, we hypothesize that H2O2 is an environmental cue that accumulates and promotes Spn biofilm formation at the transcriptional level. We will test this by examining biofilm formation and gene transcription in mutant bacteria unable to produce H2O2, following treatment of Spn with antioxidants, under anaerobic conditions or with exogenous H2O2 added, and by examining a mutant deficient in CiaRH. Work proposed in this R21 will identify the molecular mechanisms that underlay Spn biofilm formation. Knowledge obtained may identify novel targets for prevention or treatment of Spn disease. Findings made as part of the proposed work will serve as the basis of a future R01 that investigates how the identified SpxB- derived molecules drive biofilm formation.